Methods in Molecular Biology TM VOLUME 198 Neural Stem Cells Methods and Protocols Edited by Tanja Zigova, PhD Paul R Sanberg, PhD, DSc Juan R Sanchez-Ramos, PhD, MD HUMANA PRESS METHODS IN MOLECULAR BIOLOGY TM John M Walker, SERIES EDITOR 209 Transgenic Mouse Methods and Protocols, edited by Marten Hofker and Jan van Deursen, 2002 208 Peptide Nucleic Acids: Methods and Protocols, edited by Peter E Nielsen, 2002 207 Human Antibodies for Cancer Therapy: Reviews and Protocols edited by Martin Welschof and Jürgen Krauss, 2002 206 Endothelin Protocols, edited by Janet J Maguire and Anthony P Davenport, 2002 205 E coli Gene Expression Protocols, edited by Peter E Vaillancourt, 2002 204 Molecular Cytogenetics: Methods and Protocols, edited by Yao-Shan Fan, 2002 203 In Situ Detection of DNA Damage: Methods and Protocols, edited by Vladimir V Didenko, 2002 202 Thyroid Hormone Receptors: Methods and Protocols, edited by Aria Baniahmad, 2002 201 Combinatorial Library Methods and Protocols, 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and index ISBN 0-89603-964-1 (alk paper) Neurons–Laboratory manuals Stem cells–Laboratory manuals I Zigova, Tanja II Sanberg, Paul R.III Sanchez-Ramos, Juan Raymond, 1945- IV Series QP357.N473 2002 573.8'536–dc21 2001051471 Preface Over the last decade, neural stem cell research has provided penetrating insights into the plasticity and regenerative potential of the brain Stem cells have been isolated from embryonic as well as adult central nervous system (CNS) Many non-CNS mammalian tissues also contain stem cells with a more limited repertoire: the replacement of tissue-specific cells throughout the lifetime of the organism Progress has been made in understanding fundamental stem cell properties that depend on the interplay of extrinsic signaling factors with intrinsic genetic programs within critical time frames With this growing knowledge, scientists have been able to change a neural stem cell’s fate Under certain conditions, neural stem cells have been induced to differentiate into cells outside the expected neural lineage and conversely, stem cells from nonneural tissue have been shown to transdifferentiate into cells with distinct neural phenotypes At the moment, there is an accelerated effort to identify a readily available, socially acceptable stem cell that can be induced to proliferate in an undifferentiated state and that can be manipulated at will to generate diverse cells types We are on the threshold of a great new therapeutic era of cellular therapy that has as great, if not greater, potential as the current pharmacologic era, glorified by antibiotics, anesthetics, pain killers, immunosuppressants, and psychotropics Cellular therapeutics carries the promise of replacing missing neurons, but also may serve to replenish absent chemical signals, metabolites, enzymes, neurotransmitters, or other missing or defective components from the diseased or injured brain Cellular therapies may provide the best vehicle for delivery of genetic material for treatment of hereditary diseases Although a great deal of data has been gathered and insights have been provided by researchers around the world, we are still in the dark about fundamental processes that determine cell fate or that maintain a cell’s “stemness.” To take some of the mystery out of this field and to provide a practical guide for the researcher, we have collected straightforward methods and protocols used by outstanding scientists in the field Our primary goal is to facilitate research in neural stem cell biology by providing detailed protocols to both stimulate and guide novices and veterans in this area v vi Preface We divided Neural Stem Cells: Methods and Protocols into three broad sections The first section, “Isolation and Culture of Neural Stem Cells” introduces the reader to different sources of stem/progenitor cells and provides a wide range of conditions for their selection, nourishment, growth and survival in culture The second section, “Characterization of Neural Stem Cells in vitro” is a collection of the cellular, electrophysiological, and molecular techniques required to define the characteristics of neural stem cells in culture The third section, “Utilization/Characterization of Neural Stem Cells in vivo,” is a collection of techniques to identify and characterize endogenous stem cells as well as exogenous stem cells after transplantation into the brain At this stage in Neural Stem Cell Biology , we have relied on the available state-of-the-art techniques to define the properties of these cells and to test their inherent plasticity We hope that this collection of methods and protocols, ranging from simple to sophisticated in complexity, will serve as a handy guide for stem cell scientists We expect that the user will develop even more advanced techniques and strategies in this field Like a good cookbook full of recipes and cooking instructions, we are confident that experimentation with these procedures may generate even better results suited to the particular goals of the researcher We would like to acknowledge Professor John M Walker who initially suggested we put together this book and then later advised us throughout the editorial process We greatly appreciate the suggestions and encouragement from Dr Mahendra S Rao We especially thank Marcia McCall for her caring assistance, attention to detail, and long hours invested into compiling this volume Tanja Zigova, PhD Paul R Sanberg, PhD, DSc Juan R Sanchez-Ramos, PhD, MD Contents Preface v Contributors xi PART I ISOLATION AND CULTURE OF NSCS Neural Differentiation of Embryonic Stem Cells K Sue O’Shea Production and Analysis of Neurospheres from Acutely Dissociated and Postmortem CNS Specimens Eric D Laywell, Valery G Kukekov, Oleg Suslov, Tong Zheng, and Dennis A Steindler 15 Isolation of Stem and Precursor Cells from Fetal Tissue Yuan Y Wu, Tahmina Mujtaba, and Mahendra S Rao 29 Olfactory Ensheathing Cells: Isolation and Culture from the Rat Olfactory Bulb Susan C Barnett and A Jane Roskams 41 Culturing Olfactory Ensheathing Glia from the Mouse Olfactory Epithelium Edmund Au and A Jane Roskams 49 Production of Immortalized Human Neural Crest Stem Cells Seung U Kim, Eiji Nakagawa, Kozo Hatori, Atsushi Nagai, Myung A Lee, and Jung H Bang 55 Adult Rodent Spinal Cord Derived Neural Stem Cells: Isolation and Characterization Lamya S Shihabuddin 67 Preparation of Neural Progenitors from Bone Marrow and Umbilical Cord Blood Shijie Song and J Sanchez-Ramos 79 Seeding Neural Stem Cells on Scaffolds of PGA, PLA, and Their Copolymers Erin Lavik, Yang D Teng, Evan Snyder, and Robert Langer 89 vii viii Contents PART II CHARACTERIZATION OF NSCS I N VITRO A CELLULAR TECHNIQUES 10 Analysis of Cell Generation in the Telencephalic Neuroepithelium Takao Takahashi, Verne S Caviness, Jr., and Pradeep G Bhide 101 11 Clonal Analyses and Cryopreservation of Neural Stem Cell Cultures Angelo L Vescovi, Rossella Galli, and Angela Gritti 115 12 Assessing the Involvement of Telomerase in Stem Cell Biology Mark P Mattson, Peisu Zhang, and Weiming Fu 125 13 Detection of Telomerase Activity in Neural Cells Karen R Prowse 137 14 In Vitro Assays for Neural Stem Cell Differentiation Marcel M Daadi 149 15 Electron Microscopy and Lac-Z Labeling Bela Kosaras and Evan Snyder 157 B ELECTROPHYSIOLOGICAL TECHNIQUES 16 Techniques for Studying the Electrophysiology of Neurons Derived from Neural Stem/Progenitor Cells David S K Magnuson and Dante J Morassutti 179 C MOLECULAR TECHNIQUES 17 Fluorescence In Situ Hybridization Barbara A Tate and Rachel L Ostroff 189 18 RT-PCR Analyses of Differential Gene Expression in ES-Derived Neural Stem Cells Theresa E Gratsch 197 19 Differential Display: Isolation of Novel Genes Theresa E Gratsch 213 20 Cell Labeling and Gene Misexpression by Electroporation Terence J Van Raay and Michael R Stark 223 21 Gene Therapy Using Neural Stem Cells Luciano Conti and Elena Cattaneo 233 22 Modeling Brain Pathologies Using Neural Stem Cells Simonetta Sipione and Elena Cattaneo 245 Contents ix PART III UTILIZATION/CHARACTERIZATION OF NSCS I N VIVO A ENDOGENOUS POOLS OF STEM/PROGENITOR CELLS 23 Activation and Differentiation of Endogenous Neural Stem Cell Progeny in the Rat Parkinson Animal Model Marcel M Daadi 265 24 Identification of Musashi1-Positive Cells in Human Normal and Neoplastic Neuroepithelial Tissues by Immunohistochemical Methods Yonehiro Kanemura, Shin-ichi Sakakibara, and Hideyuki Okano 273 25 Identification of Newborn Cells by BrdU Labeling and Immunocytochemistry In Vivo Sanjay S P Magavi and Jeffrey D Macklis 283 26 Immunocytochemical Analysis of Neuronal Differentiation Sanjay S P Magavi and Jeffrey D Macklis 291 27 Neuroanatomical Tracing of Neuronal Projections with Fluoro-Gold Lisa A Catapano, Sanjay S P Magavi, and Jeffrey D Macklis 299 B TRANSPLANTATION 28 Labeling Stem Cells In Vitro for Identification of Their Differentiated Phenotypes After Grafting into the CNS Qi-lin Cao, Stephen M Onifer, and Scott R Whittemore 307 29 Optimizing Stem Cell Grafting into the CNS Scott R Whittemore, Y Ping Zhang, Christopher B Shields, Dante J Morassutti, and David S K Magnuson 319 30 Vision-Guided Technique for Cell Transplantation and Injection of Active Molecules into Rat and Mouse Embryos Lorenzo Magrassi 327 31 Transplantation into Neonatal Rat Brain as a Tool to Study Properties of Stem Cells Tanja Zigova and Mary B Newman 341 32 Routes of Stem Cell Administration in the Adult Rodent Alison E Willing, Svitlana Garbuzova-Davis, Paul R Sanberg, and Samuel Saporta 357 Index 375 Contributors EDMUND AU • Center for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada JUNG H BANG • Brain Disease Research Center, Ajou University School of Medicine, Suwon, Korea SUSAN C BARNETT • CRC Beatson Laboratories, Garscube Estate, Glasgow, G61 BD, Scotland PRADEEP G BHIDE • Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA QI-LIN CAO • Kentucky Spinal Cord Injury Research Center and Department of Neurological Surgery, University of Louisville, School of Medicine, Louisville, KY LISA A CATAPANO • Division of Neuroscience, Children’s Hospital, Program in Neuroscience, Harvard Medical School, Boston, MA ELENA CATTANEO • Department of Pharmacological Sciences and Center of Excellence on Neurodegenerative Diseases, University of Milan, Milan, Italy VERNE S CAVINESS • Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA LUCIANO CONTI • Department of Pharmacological Sciences and Center of Excellence on Neurodegenerative Diseases, University of Milan, Milan, Italy, and Centre for Genome Research, University of Edinburgh, Edinburgh, Scotland MARCEL M DAADI • Layton BioScience, Inc., Sunnyvale, CA WEIMING FU • Laboratory of Neurosciences, National Institute on Aging Gerontology Research Center, Baltimore, MD ROSSELLA GALLI • Institute for Stem Cell Research, Ospedale “San Raffaele,” Milan, Italy SVITLANA GARBUZOVA-DAVIS • Center for Aging and Brain Repair, Department of Neurosurgery, College of Medicine, University of South Florida, Tampa, FL THERESA E GRATSCH • Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI ANGELA GRITTI • Institute for Stem Cell Research, Ospedale “San Raffaele,” Milan, Italy KOZO HATORI • Division of Neurology, Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada xi 368 Willing et al Using blunt dissection, isolate the common, external, and internal carotid arteries from the vagus nerve and surrounding tissues in the neck Place two sutures (5-0 silk) on the external carotid, and permanently tie the one closest to the base of the skull Place the second tie next to the junction with the common carotid Do not tie Place temporary sutures or vascular clamps on the common carotid and the internal carotid to prevent blood loss once a hole is made in the external carotid Make a hole in the external carotid using a 25 gauge needle Insert a 31 gauge delivery needle into the lumen of the common carotid Tie a half knot around the needle with the suture at the junction of the external and common carotid arteries 10 Remove the clamps on the common and internal carotid 11 Inject the cells 12 Leave the needle in place for min, then remove the delivery needle 13 Completely tie the suture at the junction of the external and common carotid 14 Close the incision 3.3 Post-Operative Care For the most part, the post-operative care of the mice or rats should be fairly standard One of the most critical concerns after surgery is body temperature; anesthesia and the incision both may contribute to lowering body temperature After surgery, the first priority should be to place the animal on a heating pad and monitor it frequently Mice in particular are very sensitive to temperature changes, and are subject to both hypo- and hyper- thermia Nest building material is provided to assist mice in maintaining their body temperature and increase their sense of security In addition, the animals are placed on a prophylactic antibiotic therapy for d post surgery to minimize the risk of surgery-related infections Acetaminophen is also placed in the water for one week Finally, the animals should be monitored for dehydration Signs of dehydration include dry excrement, non-elastic skin, etc Dehydration can be remedied either by twice daily oral administration of water (1 mL per time), substitution of a soft food, or subcutaneous injections of physiological saline In addition to standard post-surgical care, transplanted animals in our care also receive immunosuppressant drugs to minimize graft rejection (Note 13) We usually use cyclosporine [10 mg/kg/day ip for rats and mice or in some cases 25 mg/kg/day orally for the mice (28)] It is not yet clear whether this treatment is necessary It can be argued that embryonic stem cells or stem cells from umbilical cord blood are non-immunogenic because they have relatively few surface markers, but it has yet to be shown that these grafts can be maintained in vivo long-term without immunosuppression Route of Cellular Administration 369 Notes After administration of Equithesin (3.5 mL/kg, ip), a rat or mouse remains anesthetized for approximately 1–1.5 h This anesthetic is generally well tolerated by the rats in particular and to a lesser extent by mice Should it be necessary to supplement the initial dose, administer 0.05 mL to the rat or 0.001–0.002 mL for a 20–23 g mouse After 3–5 min, depth of anesthesia should have increased again Respiratory problems can occur under both Equithesin and Isoflurane anesthesia These problems are more easily rectified with gas anesthesia by simply decreasing the amount of anesthetic being delivered Should problems persist with gas anesthesia or injectable anesthetic, a number of alternatives exist The simplest method is to stimulate the animal’s breathing center with smelling salts (ammonia inhalant of 15% ammonia and 35% alcohol) Three to five ammonia inhalations (each separated by 15–20 sec) should be accompanied by gentle compression of the rib cage Usually the animal is revived quickly, appearing to sneeze If there is no response, it may be necessary to administer a respiratory stimulator, such as Doxapram hydrochloride (1–2 drops under the tongue followed by 0.1 mL im) If these measures fail, it may be necessary to perform mouth to mouth resuscitation on the animal using a small tube to blow through Gentle chest compression may also help For all surgeries in which the cells are implanted directly into either the brain or spinal cord, there is an issue of seepage of the cell suspension occurring along the needle at the site of penetration This issue is usually a function of the cells being injected too quickly and can be easily remedied by injecting the cells more slowly Once the entire volume of cells has been administered, the needle should also be left in place, for striatal transplant, and up to 10 for hippocampal or cortical transplants, before being withdrawn For the cortical transplants, some basic geometry is required to calculate the insertion point and depth of insertion to reach the desired placement of the needle tip (Fig 2) The formula to determine the depth of transplantation, b, from the cortical surface, and for a given angle ⌰ is: c(cos⌰) = b Similarly, the formula to determine the lateral extent of the transplant needle tip from the cortical insertion point is: c(sin⌰) = a At times it may be easier to find the transplant target site, determine a preferred course for the needle and calculate the angle of insertion, ⌰, with the formula: sin⌰ = a/c When single bilateral injections are made in the mouse spinal cord, we prefer to hand drill burr holes through the vertebra In our hands, we get a lower incidence 370 10 11 Willing et al of hemorrhaging If multiple injections are done or we are transplanting in the rat, then we perform a laminectomy The procedure is similar through the removal of the erector spinae muscle After that, the spinous process is cut, the lamina is cut on each side, and the top of the vertebra is removed For short-term surgeries that not require a stereotaxic instrument, the gas anesthetic Isoflurane may be used The depth of anesthesia is easily maintained, and the animals recover from anesthesia faster If hemorrhaging occurs as a result of cutting the dura and pia mater, the extent of damage should be determined with a microscope or other optic magnification If the hemorrhage is extensive, surgery should be stopped and the animal must be sacrificed In the case of minimal damage, surgery can be continued The animal should be monitored extensively post-surgery to ensure that there is no surgery-related decrement in motor performance In the mouse, the femoral and tail veins are too small to practically inject the cells In the rat, it may be more appropriate to transplant an animal that has had a middle cerebral artery occlusion (MCAO) to induce stroke, not through the jugular vein but through either the femoral or tail vein The reason for this preference is that 24 h after MCAO, the neck region may be swollen and the other blood vessels around the carotid may be more reactive than they would be in a naïve animal Similarly, in an animal with a spinal cord injury that is dragging its hind limbs around the cage, a femoral artery injection may increase the risk of infection around the incision site The procedure for cannulating both veins and arteries are much the same in the rat and the mouse The main difference beyond the obvious size distinction is the consistency (strength, elasticity) of the tissue; mouse tissue is not as sturdy as rat and requires a more delicate touch One of the biggest concerns with vascular administration of the cells is not introducing an air embolus into the bloodstream Extra care must be taken in loading the syringe and needle to ensure there are no air bubbles and further, when the needle is placed in the vein or artery, the tip of the needle should be placed in the vessel so that the surgeon can see the suspension as it leaves the needle tip If, in spite of all precautions, an air bubble is injected, if the surgeon is able to see the tip and is delivering the suspension slowly, then it is possible to draw the air bubble back into the syringe In most cases, a small air bubble can be tolerated in the rat without complications, but this situation is more critical in the mouse The tail vein is largest at the base of the tail and smallest at the tip of the tail However, we choose our initial entry site (6 cm from the base), so that we can make a second or even third (rarely) attempt to enter the tail vein more rostrally should the first attempt fail Should these three attempts fail, the tail vein on the other side of the tail is also available There should be no resistance felt when depressing the plunger to deliver the cells into the tail vein A small swelling may be visible where the needle lies in the tail vein as the bolus is delivered, usually indicating that the injection is Route of Cellular Administration 371 proceeding faster than the capacity of the vein However, if a prominent swelling is seen as the injection is being made, or if resistance to depressing the plunger is felt, the needle is no longer in the vein and the injection is being made in the substance of the tail 12 The arteries are stronger and more elastic than the veins and maintain their size and shape even with extensive working The veins, on the other hand will collapse with extensive manipulation The best approach to isolating the vessels is to manipulate them only as much as is necessary to isolate them from surrounding nerves or connective tissue Even so, removing excess connective tissue from the top of the vessel is desirable since it will make it much easier to make a clean hole in the vessel and place the needle directly into the lumen If this removal is not done, the needle can end up in the connective tissue or in the wall of the vessel If, upon delivery of the cell suspension, the vessel/connective tissue bubbles up or the delivery is not smooth and easy, then the needle is not in the lumen of the vessel It may be possible to minimize this constrictive effect by applying a few drops of lidocaine before the vessel is isolated from the surrounding tissue 13 One of the most troublesome post-operative issues involves the long-term use of cyclosporin Some of the most common side effects of this treatment include dental problems that interfere with normal feeding, higher risk of opportunistic infections, and gastrointestinal problems These latter problems can range from diarrhea to bloating and constipation, and can occur in both rats and mice However, we have found that in the SOD1 mouse, in particular, the gas and constipation can be lethal within 3–5 d of onset During this time, animals are anorexic, their fur becomes disheveled, and their abdomen bloated Some researchers have reported delayed gastric emptying and delayed colonic transit time in ALS patients (29) There are also reports of small bowel bacterial overgrowth (30) that could contribute to bloating We have found some efficacy in reversing this problem through the administration of red wine (3–5 drops twice daily on the tongue of the affected mice) The mechanism underlying this effect is not yet clear, but may be related to antioxidant properties of the wine (31), modulation of intestinal motility (32), or its antibacterial activities (33) We are also exploring other pharmacological methods to speed up the transit of nutrients through the gastrointestinal tract References Vescovi, A L., Parati, E A., Gritti, A., Poulin, P., Ferrario, M., Wanke, E., Frolichsthal-Schoeller, P., Cova, L., Arcellana-Panlilio, M., Colombo, A., and Galli, R (1999) Isolation 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25 Hedreen, J C and McGrath, S (1977) Observations on labeling of neuronal cell bodies, axons, and terminals after injection of horseradish peroxidase into rat brain J Comp Neurol 176, 225–246 26 Graybiel, A M and Devor, M (1974) A microelectrophoretic delivery technique for use with horseradish peroxidase Brain Res 68, 167–173 27 Aboody, K S., Brown, A., Rainov, N G., Bower, K A., Liu, S., Yang, W., Small, J E., Herrlinger, U., Ourednik, V., Black, P M., Breakefield, X O., and Snyder, E Y (2000) From the cover: neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas Proc Natl Acad Sci USA 97, 12,846–12,851 28 Berden, J H (1986) Effects of cyclosporin A on autoimmune disease in MRL/1 and BXSB mice Scand J Immunol 24, 405–411 29 Toepfer, M., Folwaczny, C., Klauser, A., Riepl, R L., Muller-Felber, W., and Pongratz, D (1999) Gastrointestinal dysfunction in Amyotrophic lateral sclerosis Amyotrophic lateral sclerosis 1, 15–19 30 Ostermeyer-Shoaib, B., and Patten, B M (1993) IgG subclass deficiency in amyotrophic lateral sclerosis Acta Neurol Scand 87, 192–194 374 Willing et al 31 Lapidot, T., Harel, S., Akiri, B., Granit, R., and Kanner, J (1999) pH-dependent forms of red wine anthocyanins as antioxidants J Agri Food Chem 47, 67–70 32 Charles, F., Evans, D F., Castillo, F D., and Wingate, D L (1994) Daytime ingestion of alcohol alters nightime jejunal motility in man Dig Dis Sci 39, 51–58 33 Marimon, J M., Bujanda, L., Gutierrez-Stampa, M A., Cosme, A., and Arenas, J I (1998) In vitro bactericidal effect of wine against Helicobacter pylori Am J Gastro 93, 1392 Index 375 Index 112, 150, 153, 161, 168, 267, 269–270, 285-289, 309, 330, 350 administration intraparenchymal injection, 350 intraperitoneal infusion, 286 intraperitoneal injection, 105– 108, 269, 286, 350 intraventricular infusion, 287 oral ingestion 286 Buoyant density 71 A Alkaline phosphatase, 72, 191, 279, 309, 312 Alzet pump (see also Osmotic pump), 266 Antiadhesive solution, 17, 19, 24 Antibiotics, 10, 127, 256, 359 Antigen retrieval method, 274 Apomorphine test, 266 Apoptosis (see also Neuronal cell death), 247 Astrocyte monolayer, 25, 26, 47 A2B5, 30, 32, 33, 35, 36, 42, 288 Autoradiography, 105, 109, 112, 330 B β-galactosidase, 10, 162–163, 167– 168, 309 β-III-tubulin, 7, 9, 32, 33, 35, 36, 56, 58, 63, 81, 150, 315 Bone marrow (bone marrow stromal cells, BMSC), 79–87 co-culture of bone marrow with fetal midbrain cells, 85 isolation, 82–84 neural induction in vitro, 85 Bone morphogenetic protein (BMP), 9, 11, Brain-derived neurotrophic factor (BDNF), 5, 79, 84, 152 Bromodeoxyuridine (BrdU, BUdR, BrdUrd), 104–105, 108–109, C Cell generation, 101–112 proliferation, 101–112 viability, 323–324, 325, 343, 346, 347–348, 353 Chick embryo extract (CEE), 29, 33, 38, 39 Cholera toxin B, 360–361, Chromogranin, 56, 58, 63 Ciliary neurotrophic factor, 152 Clonal analysis, 115–122 limiting dilution, 116-119 methylcellulose assay, 117, 119– 120 subcloning procedure, 117, 120 Colcemid, 57, 61, Cryopreservation, 115–122 D Desmin, 56, 58, 63, Differential display (DD), 213–221 375 376 cloning DD-PCR (polymerase chain reaction) products/ cDNAs, 215, 220 differential display PCR (DDPCR), 214, 216, DD-PCR sequencing gel electrophoresis, 214, 217 Northern/reverse Northern hybridizations, 215, 219–220 reamplification of DD-PCR products, 214, 217–219 reverse transcription reaction (DD-RT), 214, 216 RNA extraction and purification, 214–216 Differential gene expression, 197–211 polymerase chain reaction, 198– 199, 202–203 purification and cloning of PCR products, 199, 204–207 reverse Northern blots, 199, 207–211 reverse transcription, 198, 201–202 RNA extraction and purification, 198–201 DiI (fluorescent dye, 1,1′- dioctadecyl -3,3,3′,3′-tetramethylindocyanine perchlorate), 308–309, 329, 360–361 Dimethyl sulfoxide (DMSO), 6, 31, 69, 73, 85, 211, 239, 261, 359 Dispase, 38 Doublecortin (Dcx, Migratory neuronal marker), 288, 291–296 E Epidermal growth factor (EGF, see Growth factors) Electron microscopy (EM, see also LacZ), 157–172 Index β-gal immunohistochemistry, 162–163, 167–168 tissue preservation and processing for EM, 163– 164, 166, 168–172 transfection of cells with the reporter bacterial lac-Z gene, 161–162, 164–165 X-gal histochemical reaction, 162, 165–167, Electrophysiology, 179–186 recordings from cultured neural stem cells (NSCs), 181, 183–186 recordings from slice preparations, 180–181, 182–183 recordings from transplanted NSCs, 180–183 Electroporation, 223–231 apparatus, 223, 226 DNA injection, 226 expression plasmid, 224 in ovo, chick embryo, 224–225, 227–228 in vitro, embryonic neural tube, 225–226, 228–231 Embryoid body, Embryonic stem (ES) cells, 3–12 ES cell culture 4–5, 6–7 growth medium, neural differentiation, 5, 7–8, neuronal differentiation, 9–10 Endogenous pools of NSCs, in general, activation of NSCs, 265–271 differentiation of NSCs, 265–271, 291–297 identification of newborn cells, 101–112, 283–290 Index Musashi-1 immunohistochemistry, 273–281 neuroanatomical tracing of neuronal projections (see also fluorogold), 299–304 Engineering growth-factor expanded NSCs, 240 immortalized NSC lines, 241 Enhanced green fluorescent protein (EGFP, reporter gene), 309–310, 316, 331 Enzymatic separation, 30, 31, 70 Epithelium, pseudostratified ventricular (PVE), 101, 103, 110 estimation of P and Q fractions, 105–108 growth modes, 103–104, Ethidium bromide, 57, 61, 126, 130, 201, 248, 251 Exponential expansion, 103 F FACS (see Fluorescence activated cell sorting) Fibronectin, 10, 30, 36, 81, 184 Fluorescence-activated cell sorting (FACS), 45–46, 140, 312 FISH (see fluorescence in situ hybridization) Fluorescein diacetate/propidium iodide (see Cell viability), 343, 347–348 Fluorescence in situ hybridization (FISH), 189–194 in situ hybridization, 193–194 preparation of probes by DIG labeling, 193 preparation of tissue, 192–193 377 probes, 190–192 Fluorogold (FG, retrograde marker), 299–303 G Galactocerebroside, anti GalC, 42, 45, 46, 150 Gene misexpression (see electroporation) Giemsa stain, 57, 61 Glial fibrillary acidic protein (GFAP, an astrocytic marker), 9, 42, 47, 49, 51, 53, 63, 79, 81, 150, 151, 278, 288, 315 Glial-restricted precursors (GRP), 30 Green fluorescent protein (GFP) transgenic mouse, 10, 21, 72, 155, 223, 229, 231 Growth factors basic fibroblast growth factor (bFGF), 5, 7, 9, 30, 35, 56, 57, 59, 68, 72, 73, 117, 120, 150, 152, 266–268 epidermal growth factor (EGF), 57, 59, 79, 117, 120, 150, 152, 266–269 nerve growth factor (NGF), 57 platelet-derived growth factor, 31, 36 stock solution 17, 19, 31, Guanidine cyanide method, 22 H Hoechst dye 33258, bisbenzimide, DAPI, Feulgen, nuclear stain, 285, 293, 295–296, 309 33342, 2′-(4-ethoxyphenyl)-5-(4methyl-1-piperazinyl)-2.5′- 378 bi-1H-benzimidazole), 309, 330-331 hNT cells (see also NT2N cells), 343, 346, 353, 361 hTERT (see also TERT), 125 Hu (early neuronal marker, RNAbinding protein), 81, 275, 276, 288, 291-296 Hyaluronidase 39, 51 I Immortalization f human neural crest stem cells (see also Neural crest stem cells), 61 of NSCs, 239-240 growth factor-expanded, 240 retroviral mediated, 239 Immunopanning, 30, 36–39 Iododeoxyuridine (IrdU, halogenated thymidine analog), 284 Isolation of stem and precursor cells from fetal tissue, 29–39 dissociation and plating of neuroepithelial (NEP) cells, 30, 33, 34 freezing NEP cells, 34, 35 maintenance and testing of undifferentiated NEP cells, 30, 33, 35 passaging NEP cells, 30–31, 33– 34, 35, 39 removal of neural tubes, 33 Index membrane (see also DiI, PKH26), 309 nuclear (see also Hoechst, BrdU, 3H-thymidine), 309 reporter genes (see also lacZ, EGFP, enhanced green fluorescent protein), 309 LacZ (reporter gene), 72, 161–163, 165–168, 309, 311–313 Laminin, 5, 6, 8, 10, 11, 17, 31, 36, 68, 118, 184 Leukemia inhibitory factor (LIF), 5, 6, 7, 10 Low affinity nerve growth factor receptor (LNGFR, p75), 42, 47, 56, 57, 63 K Karyotype, 61, 62, M Mechanical dissection, 30, 70 Medium serum-free chemically defined, 59 Microtubule-associated protein (MAP2), 58, 63, 81, 315 Musashi-1 (RNA-binding protein, marker for NSCs or progenitor cells), 81 immunohistochemistry double immunostaining with neuronal or glial markers, 278–279 prestaining treatment, 276–277 staining procedure, 277 Migratory patterns, 110–112 MTT assay (see also Neuronal death), 95 Myosin, 63 L Labeling cytoplasmic, 309 N NEP basal medium, 30, 226 Nestin, 4, 5, 32, 33, 56 Index Neural crest stem cells, 55–64 cytogenetic analysis, 57, 61 dorsal root ganglion cell culture, 57, 58–59 generation of immortalized human neural crest stem cells, 61 immunocytochemical staining for specific markers, 57, 61–64 retroviral-mediated gene transfer, 59–61 retroviral vector, 57 Neural determination genes neuroD3, 4, 197 developmental control genes myo D, neuro DS, Sox2, induction genes, noggin, 4, 197 Neural stem cells co-culture with astrocytes, 153–154 induction of tyrosine hydroxylase expression, 154-155 in vitro assays for differentiation, 149–155 Neuroanatomical tracing (see Fluorogold) Neuroepithelial cells (NEP), 29 Neuroepithelium, telencephalic, 101 Neurofilament proteins, 56, 58, 81, 315 Neuronal cell death (assays, markers) DNA laddering 250–251, 259–260 electronic cell counting, 250, 258 MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide), 95, 250, 258–259, 261 Neuron-restricted precursors (NRP), 29 Neurospheres, 8–9, 15–26, generation from acutely dissociated subependymal zone, 16–17, 18–20 379 gene analysis, 18, 22–23 false, 23 immunolabeling, 17, 20–21 postmortem tissue as a source, 16 ultrastructural analysis, 17–18, 21–22 Neurotrophin-3 (NT-3), 9, 30, 31, 35, 36 NT2 cells (human teratocarcinoma cell line, precursor cells for NT2N neurons, hNTs), 361 NT2N cells (neurons derived from NT2/D1 clone through retinoic acid exposure, also called hNT cells), 361 O O4 (oligodendrocyte marker), 42, 47, 288, 315 Olfactory ensheating glia (OEG), 49–54 cytotoxic elimination of fibroblasts, 51, 53 dissection of the olfactory epithelium, 50–52 single cell suspension, 51–52 verification of OEG phenotype, 51, 53 Olfactory ensheating cells (OEC), 41–47 dissection and degradation of olfactory bulb, 44–45 FACS purification of OEC, 45–47 preparation of astrocyte conditioned media, 42–44 purification of OEC from the olfactory bulb, 42 Osmotic pump (Alzet) implantation, 266, 268–270 preparation, 266, 268 380 P Papain, 38 Parkinson animal model, 265–266 Peripherin, 58 PKH26, fluorescent dye, 85, 86, 309, 329–330, 343, 348–350 Poly-L-lysine 31, 33, 36, 42, 43, 52, 53, 57, 62 Poly-ornithine 5, 8, 9, 17, 68, 69, 72, 73, 74, 184 Polysialic acid NCAM (E-NCAM, cell surface antigen), 30, 31, 32, 35, 36 R Retrovirus-mediated gene transfer, 59–61 Rosa 26 (see also Transplantation, other labeling strategies, Transgenic animals), 10, 155, 331–332 S Scaffolds, degradable, 89–96 polyglycolic acid (PGA), polylactic acid (PLA) and their copolymers poly(lactic-coglycolic acid, PLGA), 89–91 seeding static, 93–94 dynamic, 94–95 sterilization, 93 storage, 92–93 Serum-free media components, 152 S-100, 49, 53, 56, 58, 63, 81 Soybean trypsin inhibitor, 38, 42 S-phase labeling methods (see also bromodeoxyuridine, tritiated thymidine), 101–112 Index Spinal cord derived neural stem cells 67–76 differentiation, 74 enzymatic digestion, 70–71 immunocytochemical analysis, 74–75 isolation, 68, 72–73 passaging, freezing, reculturing, 68, 73–74 Percoll gradient purification, 71 tissue dissection, 69–70 Synthetic materials (see scaffolds) T Telomerase activity assays, 125–134 Telomerase assays, 137–147 standard TRAP (telomeric repeat amplification protocol) assay, 140, 142–144 TRAP assay for single cells or small numbers of cells, 140– 141, 144–147 TERT (telomere reverse transcriptase), 125 cellular localization of TERT protein-immunostaining, 127, 131–132 immunoblot analysis, 127, 130–131 overexpression of TERT, 127,132 RT-PCR analyses of TERT and telomere-associated protein mRNA levels, 126, 128–130 suppression of TERT expression and activity, 127, 132–134 Tetracycline-based regulatory system, 233–235 Transfection of packaging cell line, 236–239 Transplantation of NSCs Index brain target sites adult brain, 357–374 embryonic brain, 327–340 neonatal brain, 341–356 NSCs labeling identification after grafting, 307–318 in vitro, 308–311, 329–330, 348–350 with reporter genes, 311–313, 331 other strategies (allogeneic grafts, gender-specific grafts, transgenic animals, xenografts), 313–314, 331–332 optimizing NSCs grafting, 319–326 cell number and viability, 323– 324, 347–348 engraftment parameters, 324–325 injection system, 322–323 micropipet preparation, 320–322 routes of NSCs delivery intraparenchymal cortex, 363 hippocampus, 362–363 spinal cord, 364–365 striatum, 351, 360–362 subventricular zone, 351 vascular 381 arterial, 367–368 femoral vein, 366–367 jugular vein, 366 tail vein, 365–366 transuterine, 332–333 post transplantation care neonates, 351-353 adults, 359–360, 368–371 TRAP (see Telomerase assays) Tritiated thymidine, 3H-TdR, 102– 103, 105–108, 110–112, 309, 310, 311, 330 Trypan blue (see also Cell viability), 117, 118, 319, 323–324, 333, 335, 346, 360 Trypsin/EDTA solution, 5, 6, 9, 10, 11, 17, 18, 24, 26, 31, 33, 34, 35, 43, 51, 68, 72, 73, 84, 153, 230, 247, 249, 250, 256, 342 Tyrosine hydroxylase (TH), 150, 154–155, 266–268 U Umbilical cord blood (UCB) cells, 84–85 V Vimentin, 9, 56 Y Y chromosome, 191, 313 METHODS IN MOLECULAR BIOLOGY • 198 TM Series Editor: John M.Walker Neural Stem Cells Methods and Protocols Edited by Tanja Zigova, PhD and Paul R Sanberg, PhD, DSc Department of Neurosurgery, University of South Florida, College of Medicine, Tampa, FL Juan R Sanchez-Ramos, PhD, MD Department of Neurology, University of South Florida, College of Medicine, Tampa, FL Over the last decade, advances in neural stem cell research have raised the hope that one day cellular therapy will not only replace missing neurons, but also replenish absent chemical signals, metabolites, enzymes, neurotransmitters, or other missing or defective components from the diseased or injured brain In Neural Stem Cells: Methods and Protocols, internationally recognized experts from academic, clinical, and pharmaceutical laboratories describe in great detail the most frequently used cellular, molecular, and electrophysiological methods to isolate, characterize, and utilize neural stem cells These readily reproducible techniques introduce the various sources of stem/progenitor cells, provide a wide range of conditions for their culture, and make it possible to define their properties in culture Additional techniques are designed to help researchers identify endogenous stem cells as well as exogenous stem cells after transplantation in the brain The protocols range from the simplest methods of isolation and characterization of neural cell properties to such very sophisticated methods as characterizing gene expression, telomerase assays, and cell cycle kinetics Each is written by an investigator who has used the method extensively, and includes step-by-step instructions, tips on avoiding pitfalls, and invaluable notes that make all the difference to successful experimental outcomes Comprehensive and easy-to-follow, Neural Stem Cells: Methods and Protocols provides a powerful synthesis of today’s key in vitro and in vivo techniques to identify and characterize neural stem cells for research and developing experimental therapeutics FEATURES • Synthesis of latest techniques for isolation, identification, and characterization of neural stem cells • Cellular techniques, including cell cycle kinetics, telomerase assays, and electrophysiology • Molecular methods for in situ hybridization, RT-PCR analysis, gene expression, and differential display of novel genes • Extensive current bibliography CONTENTS Part I Isolation and Culture of NSCs Part II Characterization of NSCs In Vitro A Cellular Techniques B Electrophysiological Techniques C Molecular Techniques Part III Utilization/Characterization of NSCs In Vivo A Endogenous Pools of Stem/Progenitor Cells B Transplantation 90000 Methods in Molecular BiologyTM • 198 NEURAL STEM CELLS METHODS AND PROTOCOLS ISBN: 0-89603-964-1 humanapress.com 780896 039643 ... Methylation Protocols, edited by Ken I Mills and Bernie H, Ramsahoye, 2002 199 Liposome Methods and Protocols, edited by Subhash C Basu and Manju Basu, 2002 198 Neural Stem Cells: Methods and Protocols, ... research in neural stem cell biology by providing detailed protocols to both stimulate and guide novices and veterans in this area v vi Preface We divided Neural Stem Cells: Methods and Protocols. .. Aquino de Muro and Ralph Rapley, 2002 178.`Antibody Phage Display: Methods and Protocols, edited by Philippa M O’Brien and Robert Aitken, 2001 177 Two-Hybrid Systems: Methods and Protocols, edited